Thr4 phosphorylation on RNA Pol II occurs at early transcription regulating 3′-end processing

RNA polymerase II relies on a repetitive sequence domain (YSPTSPS) within its largest subunit to orchestrate transcription. While phosphorylation on serine-2/serine-5 of the carboxyl-terminal heptad repeats is well established, threonine-4’s role remains enigmatic. Paradoxically, threonine-4 phosphorylation was only detected after transcription end sites despite functionally implicated in pausing, elongation, termination, and messenger RNA processing. Our investigation revealed that threonine-4 phosphorylation detection was obstructed by flanking serine-5 phosphorylation at the onset of transcription, which can be removed selectively. Subsequent proteomic analyses identified many proteins recruited to transcription via threonine-4 phosphorylation, which previously were attributed to serine-2. Loss of threonine-4 phosphorylation greatly reduces serine-2 phosphorylation, revealing a cross-talk between the two marks. Last, the function analysis of the threonine-4 phosphorylation highlighted its role in alternative 3′-end processing within pro-proliferative genes. Our findings unveil the true genomic location of this evolutionarily conserved phosphorylation mark and prompt a reassessment of functional assignments of the carboxyl-terminal domain.

The PDF file includes: Figs. S1 to S6 Tables S6 and S7 Legends for tables S1 to S5 Other Supplementary Material for this manuscript includes the following: Tables S1 to S5 Fig. S1.Characterization of pThr4 antibody (clone 6D7) for ChIP analysis.analysis on mRNA-seq data from HEK293T cells expressing T4A or WT CTD.The table displays the defined five types of splicing events and the total number of significant events for each type (FDR <0.05 and ILD>=10%) (G) GO analysis of biological processes for transcripts with significant exon inclusion and exclusion events.p < 0.001 (**) and p < 0.0001 (***).

Molprobity score
2.24 α / 88th percentile β *Values for the corresponding parameters in the outermost shell in parenthesis.ϒ CC1/2 is the Pearson correlation coefficient for a random half of the data, the two numbers represent the lowest and highest resolution shell respectively.± Rfree is the Rwork calculated for about 10% of the reflections randomly selected and omitted from refinement.α The Molprobity score is calculated by combining the Clashscore with rotamer and Ramachandran percentage and scaled on the basis of X-ray resolution.β The 100 th percentile is considered the best and the 0 th percentile as the worst among structures of similar resolution.^There is only one Ramachandran outlier, which corresponds to Lys 48 on chain B with strong electron density (A) Dot blot showing serial dilutions of a biotin-pSer2 , biotin-pY1, biotin-pSer5, and biotin-pSer7 CTD peptide incubated with pThr4 antibody for cross-reactivity analysis.(B) Dot blot showing singly pT4 peptide substrate treated with either Abl1 or Dyrk1a kinase and incubated with either pTyr1 or pSer2 antibody.(C) Dot blot showing serial dilutions Dyrk1a-treated pT4 CTD peptide vs no-kinase treated pT4 control peptide, and Abl1-treated pT4 vs no-kinase treated pT4 control peptide containing either 10μg or 5μg of peptide.Comparison analysis for all dot blots were performed using unpaired t-test.(D) Structural model of a pSer2 or pThr4 CTD peptide positioned in the active site of Ssu72 with steric clashes around Tyr1 shown.(E) Western blot showing pSer2 recognition in HEK293 cell lysate treated with Ssu72 or catalytically inactive Ssu72.Comparison was performed using unpaired (Ssu72 WT vs untreated) or paired (Ssu72 WT vs Ssu72 Mut) t-tests.In all plots, mean with SEM are shown and quantification was from three independent biological replicates.

Fig. S2 .
Fig. S2.ChIP-seq analysis of pThr4 Pol II samples treated with Ssu72.(A) Dot blot showing a time course of chromatin isolated from HEK293T cells treated with Ssu72 for 30, 60 or 90 minutes and incubated with pThr4 antibody to detect a suitable reaction time for ChIP-seq (B) Dot blot showing a dose course of chromatin treated with Ssu72 at different concentrations and incubated with pThr4 antibody.Comparison was performed with unpaired t-test.(C) A binding affinity heatmap of 6 biological replicates of pThr4 ChIP-seq samples treated with Ssu72.FriP (Fraction of Reads in Peaks) scores are listed in the table.(D) ChIP-qPCR of gene regions enriched with pThr4 Pol II treated with Ssu72 WT compared to Ssu72 mutant.Shown are mean with SEM from 2-3 independent immunoprecipitations.Statistical comparison was performed using one-tailed t-test.(E) Comparison analysis of pThr4ChIP without Ssu72 or with Ssu72.Unpaired one-tailed t-test was applied to the two biological replicates (reps1-2) to compare TSS/TES ratio of pThr4 ChIP signal.The y-axis corresponds to log2 of the ratio of the normalized counts between IP and input samples.TSS/TES values are

Fig. S5 .
Fig. S5.Quality Control of RPRD1B ChIP-seq data.(A) Expression of HA-RPRD1B in HEK293 cells (50 μg loaded) used for ChIP-seq.(B) Scatter plots showing the pearson correlation between ChIP-seq replicate datasets of HA-RPRD1Bbound regions.The genome was divided into bins of 10 kb and the number of mapped reads in the individual bins was calculated.(C) ChIP-qPCR analysis of peaks at promoter sites of selected genes from three biological replicates of HA-RPRD1B ChIP samples and IgG control samples.Fold enrichment was calculated by comparing the positive locus sequence in ChIP DNA over the negative IgG sample.For each data point, n = 3, error bars indicate standard deviation of three biological replicates.Comparison analysis was performed using unpaired t-

Table S2 .
MS proteomic data

Table S4 .
Alternative polyadenylation events

Table S5 .
Alternative splicing events